1. Field of the Invention
This invention relates generally to the receiving and dispensing of samples such as in the field of separation of biomolecules and, in particular, separations by capillary electrophoresis and the use of the capillary electrophoresis to detect such biomolecules.
In a range of technology-based business sectors, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time-consuming, and may be entirely impracticable where only a very small quantity of a key sample or component of the analysis is available, or where a component is very costly.
Accordingly, considerable resources have been directed to developing methods for high-throughput chemical synthesis, screening, and analysis. Subsequently, considerable art has emerged, in part, from such efforts. Automated laboratory workstations have contributed significantly to advances in pharmaceutical drug discovery and genomics over the past decade. See for example, U.S. Pat. Nos. 5,104,621 and 5,356,525 (Beckman Instruments). More specifically, robotics technology has played a major role in providing a practical useful means for enabling high throughput screening (AS) methods. Reference can be made to U.S. Pat. No. 4,965,049.
In addition to the emergence of automation technology, the last decade has seen an enormous advance in the scientific understanding of critical cellular processes, and this has led to rationally designed approaches in drug discovery. Also, the application of molecular genetics and recombinant DNA technology, U. S. Pat. No. 4,237,224 (Cohen and Boyer), has led to the isolation of many genes encoding proteins which show promise as targets for new drugs. Once a target gene is identified, the recombinant protein can be heterologously expressed in mammalian tissue culture cells, insect cells, bacteria and/or yeast.
The advantages of employing molecular cloning methodologies techniques are many. Often receptors and enzymes exist in alternative forms, subtypes or isoforms. Using a cloned target focuses the primary screen on the subtype appropriate for the disease. Agonists or antagonists can be identified and their selectivity can then be tested against the other known subtypes. The availability of such cloned genes and corresponding expression systems have enabled new types of screens to be created that are specific, sensitive, and often automatable. Matched with the scientific and technological advances in biology has been the emergence of innovative methods for highly parallel chemical synthesis. For several decades, preparation of synthetic analogs to the prototypic lead compound was the established method for drug discovery. Natural products were usually isolated from soil microbes and cultured under a wide variety of conditions. The spectrum of organisms employed by the pharmaceutical industry for isolation of natural products has now expanded from actinomycetes and fungi to include plant, marine organisms, and insects
During the last five years, the chemistry of creating combinatorial libraries has made a vastly increased number of synthetic compounds available for testing. More specifically, thousands to tens or hundreds of thousands of small molecules can be rapidly and economically synthesized. See, for example, the combinatorial chemistry patents (U.S. Pat. No. 5,252,743 (Affymax Technologies N.V.). Thus, combinatorial libraries complement the large numbers of synthetic compounds available from the more traditional drug discovery programs based, in part, on identifying lead compounds through natural product screening.
Competitive binding assays, originally developed in the 1960's for immunodiagnostic applications, continue to be commonly employed for quantitatively characterizing receptor-ligand interactions. Despite advances in the development of spectrophotometric and fluorometric-based bioanalytical assays, radiolabeled ligands are still commonly employed in pharmaceutical HTS applications. Although non-isotopic markers promise to be environmentally cleaner, safer, less expensive, and generally easier to use than radioactive compounds, sensitivity limitations have prevented these new methods from becoming widespread. Another major disadvantage of the competition assay is the number of steps, most notably, washing steps, required to run the screen.
A few years ago, Scintillation Proximity Assays were introduced by Amersham and also are discussed in U.S. Pat. Nos. 4,271,139 and 4,382,074 as a means of circumventing the wash steps required in the above heterogeneous assays The new homogeneous assay technology, which requires no separation of bound from free ligand, is based on the coating of scintillant beads with an acceptor molecule, for example, the target receptor.
Another variation of this theme avoids the use of radioactivity and is especially useful in high-throughput assays The modification involves the use of lanthanide chelates in time-resolved fluorometry. Aspects of this particular homogeneous assay technology are discussed in U.S. Pat. No. 5,637,509. This particular technology takes advantage of the unique properties of the lanthanide chelate europium-cryptate in combination with the energy-absorbing molecule, allophycocyanin (APC).
Robotic-based high-throughput tools are now routinely used for screening libraries of compounds for the purpose of identifying lead molecules for their therapeutic potential. Subsequently, considerable art has emerged. For example, PerSeptive Biosystems' screening method for characterizing ligand binding to a given target employs a variety of separation techniques and is described filer in the PCT application WO 97/01755. Another related method is described in U.S. Pat. No. 5,585,277 (Scriptgen Pharmaceuticals).
Highly parallel and automated methods for DNA synthesis and sequencing have also contributed significantly to the success of the human genome project to date. For DNA synthesis instrumentation, see, e.g., PE/Applied Biosystems (ABI), PerSeptive BioSystems, Pharmacia Biotech, and Beckman Instruments. For DNA sequencing, see, e.g., ABI and LiCor. In addition, see U.S. Pat. No. 5,455,008. For a related invention, see Genzyme Corporation's HTS method for DNA analysis that is described in U.S. Pat. No. 5,589,330. For sequencing by hybridization, see PCT WO 89/10977 (Southern), Affymetrix (U.S. Pat. Nos. 5,599,695 and 5,631,734), and U.S. Pat. No. 5,202,231 (Drmanac, et al.).
Computerized data handling and analysis systems have also emerged with the commercial availability of high-throughput instrumentation for numerous life sciences research and development applications. Commercial software, including database and data management software, has become routine in order to efficiently handle the large amount of data being generated. Bioinformatics has emerged as an important field.
With the developments outlined above in molecular and cellular biology, combined with advancements in combinatorial chemistry, have come an exponential increase in the number of targets and compounds available for screening. In addition, many new genes and their expressed proteins will be identified by the Human Genome project and will therefore greatly expand the pool of new targets for drug discovery. Subsequently, an unprecedented interest has arisen in the development of more efficient ultra-high throughput methods and instrumentation for pharmaceutical and genomics screening applications. In recent parallel technological developments, miniaturization of chemical analysis systems, employing semiconductor processing methods, including photolithography and other wafer fabrication techniques borrowed from the microelectronics industry, has attracted increasing attention and has progressed rapidly. The so-called "lab-chip" technology enables sample preparation and analysis to be carried out onboard microfluidic-based cassettes. Moving fluids through a network of interconnecting enclosed microchannels of capillary dimensions is possible using electrokinetic transport methods.
Application of microfluidics technology embodied in the form of analytical devices has many attractive features for pharmaceutical high throughput screening. Advantages of miniaturization include greatly increased throughput and reduced costs, in addition to low consumption of both sample and reagents and system portability. Implementation of these developments in microfluidics and laboratory automation holds great promise for contributing to advancements in life sciences research and development.
Nonetheless, the 96 well microtiter plate has and still is the pharmaceutical industry standard for carrying out bioanalytical assays despite the recent advances in miniaturization and microfluidics. Because an enormous number of synthetic libraries have and continue to be generated using this particular multiwell format, the microtiter plate will remain entrenched within the industry.
Automated workstations for drug discovery and genomics applications are not capable of incorporating microfluidic multi-assay cards into existing robotic based high throughput microtiter plate handling and assay systems. Thus, as microfluidic technologies advance, new methods for enabling fluid transfer between multi-well plates and microassay cassettes would be beneficial. A critical factor currently limiting such a microfluidic HTS hybrid device is a means for reproducible liquid communication between the disparate dimensions of the two systems. More specifically, integration of microfluidics technology with existing robotic-based methods currently used in automated workstations is constrained by differences in volume size of samples used. For these reasons, new automated methods for multiplexing common lab tasks such as sample handling and dispensing on the microscale is required.
Capillary-based separations are widely used for analysis of a variety of analyte species. Numerous subtechniques, all based on electrokinetic-driven separations, have been developed. Capillary electrophoresis is one of the more popular of these techniques and can be considered to encompass a number of related separation techniques such as capillary zone electrophoresis, capillary gel electrophoresis, capillary isoelectric focusing, capillary isotachophoresis, and micellar electrokinetic chromatography. In the context used throughout this application, the phrase "capillary electrophoresis" is used to refer to any and all of the aforementioned electrokinetic separation subtechniques.
Electrophoresis is a separation process in which molecules with a net charge migrate through a medium under the influence of an electric field. Traditionally, slab gel electrophoresis has been a widely used tool in the analysis of genetic materials. See, for example, G. L. Trainor, Anal. Chem. (1990) 62:418-26. Capillary electrophoresis has emerged as a powerful separation technique with applicability to a wide range of molecules from simple atomic ions to large DNA fragments. In particular, capillary electrophoresis has become an attractive alternative to slab electrophoresis for biomolecule analysis, including DNA sequencing. See, for example, Y. Baba, et al., Trends in Anal. Chem. (1992) 11:280-287. This is generally because the small size of the capillary greatly reduces Joule heating associated with the applied electrical potential. Furthermore, capillary electrophoresis requires less sample and produces faster and better separations than slab gels.
Currently, sophisticated experiments in chemistry and biology, particularly molecular biology, involve evaluating large numbers of samples. For example, DNA sequencing of genes is time consuming and labor intensive. In the mapping of the human genome, a researcher must be able to process a large number of samples on a daily basis. If capillary Enelectrophoresis can be conducted and monitored simultaneously on many capillaries, i.e., multiplexed, the cost and labor for such projects can be significantly reduced. Attempts have been made to sequence DNA in slab gels with multiple lanes to achieve multiplexing. However, slab gels are not readily amenable to a high degree of multiplexing and automation.
Difficulties exist in preparing uniform gels over a large area, maintaining gel to gel reproducibility and loading sample wells. Furthermore, difficulties arise as a result of the large physical size of the separation medium, the requirements for uniform cooling, large amounts of media, buffer, and samples, and long run times for extended reading of nucleotide sequences. Unless capillary electrophoresis can be highly multiplexed and multiple capillaries run in parallel, the advantages of capillary electrophoresis cannot produce substantial improvement in shortening the time needed for sequencing the human genome.
Capillary electrophoresis possesses several characteristics which makes it amenable to this application. The substantial reduction of Joule heating per lane makes the overall cooling and electrical requirements more manageable. The cost of materials per lane is reduced because of the smaller sample sizes. The reduced band dimensions are ideal for excitation by laser beams, as well as focused broad band sources, and for imaging onto array detectors or discrete spot detectors. The concentration of analyte into such small bands results in high sensitivity. The use of electromigration injection, i.e., applying the sample to the capillary by an electrical field, provides reproducible sample introduction with little band spreading, minimal sample consumption, and little labor.
Among the techniques used for detecting target species in capillary electrophoresis, laser-excited fluorescence detection so far has provided the lowest detection limits. Therefore, fluorescence detection has been used for the detection of a variety of analyses, especially macromolecules, in capillary electrophoresis. There have been attempts to implement the analysis of more than one capillary simultaneously in the electrophoresis system, but the number of capillaries has been quite small. For example, S. Takahashi, et al., Proceedings of Capillary Electrophoresis Symposium, December, 1992, referred to a multicapillary electrophoresis system in which DNA fragment samples were analyzed by laser irradiation causing fluorescence. This method, however, relies on a relatively poor focus (large focal spot) to allow coupling to only a few capillaries. Thus, this method could not be applied to a large number of capillaries. This method also results in relatively low intensity and, thus, poor sensitivity because of the large beam. Furthermore, detection in one capillary can be influenced by light absorption in the adjacent capillaries, thus affecting accuracy due to cross-talk between adjacent capillaries.
Attempts have been made to perform parallel DNA sequencing runs in a set of up to 24 capillaries by providing laser-excited fluorometric detection and coupling a confocal illumination geometry to a single laser beam and a single photomultiplier tube. See, for example, X. C. Huang, et al., Anal. Chem. (1992) 64:967-972, and Anal. Chem. (1992) 64:2149-2154. Also see U.S. Pat. No. 5,274,240. However, observation is done one capillary at a time and the capillary bundle is translated across the excitation/detection region at 20 mm/see by a mechanical stage.
There are features inherent in the confocal excitation scheme that limit its use for very large numbers of capillaries. Because data acquisition is sequential and not truly parallel, the ultimate sequencing speed is generally determined by the observation time needed per DNA band for an adequate signal-noise ratio. Moreover, the use of a translational stage can become problematic for a large capillary array. Because of the need for translational movement, the amount of cycling and therefore bending of the capillaries naturally increases with the number in the array. It has been shown that bending of the capillaries can result in loss in the separation efficiency. This is attributed to distortions in the gel and multipath effects. Sensitive laser-excited fluorescence detection also requires careful alignment both in excitation and in light collection to provide for efficient coupling with the small inside diameter of the capillary and discrimination of stray light. The translational movement of the capillaries thus has to maintain stability to the order of the confocal parameter (around 25 .about.m) or else the cylindrical capillary walls will distort the spatially selected image due to misalignment of the capillaries in relation to the light source and photodetector. In addition, long capillaries provide slow separation, foul easily, and are difficult to replace.
2. Previous Disclosures
U.S. Pat. No. 5,324,401 to Young, et al., describes a multiplexed capillary electrophoresis system where excitation light is introduced through an optical fiber inserted into the capillary. In this system the capillaries remain in place, i.e. in the buffer solutions when the capillaries are read.
U.S. Pat. No. 5,332,480 (Datta, et al.) describes a multiple capillary electrophoresis device for continuous batch electrophoresis.
U.S. Pat. No. 5,277,780 (Kambara) describes a two dimensional capillary electrophoresis apparatus for use with a two dimensional array of capillaries for measuring samples, such as DNA samples, in an array of test wells.
U.S. Pat. 5,413,686 (Klein and Miller) describes a multi-channel automated capillary electrophoresis analyzer in which multiple individual separation capillaries are installed in a instrumental analyzer which serves to flush and fill the capillaries and associated buffer reservoirs from supplies of buffer situated within the instrument.
U.S. Pat. 5,439,578 (Dovichi and Zhang) describes a multiple capillary biochemical analyzer based on an array of separation capillaries terminating in a sheath flow cuvette. The use of the sheath flow cuvette facilitates detection of the analyte bands by reducing the magnitude of scattered radiation from the detection zone.
U.S. Pat. No. 5,338,427 (Shartle, et al.) describes a single use capillary cartridge having electrically conductive films as electrodes; the system does not provide for multiplexed sampling, sample handling, and electrophoresis.
U.S. Pat. Nos. 5,091,652 (Mathies, et al.) and 4,675,300 (Zare, et al.) describe means for detecting samples in a capillary.
U.S. Pat. No. 5,372,695 (Demorest) describes a system for delivering reagents to serve a fix capillary scanner system.
Numerous examples of sample handling for capillary electrophoresis are known. For example, James in U.S. Pat. No. 5,286,652 and Christianson in U.S. Pat. No. 5,171,531 are based on presenting a single vial of sample to a single separation capillary for a sequential series of analyses.
Goodale in U.S. Pat. No. 5,356,525 describes a device for presentation of a tray of 7 vials of samples to an array of seven capillaries for the sample injection process.
Carson in U.S. Pat. No. 5,120,414 describes injection of a sample contained within a porous membrane onto a single capillary electrophoresis device. The end of the capillary must be in intimate contact with the porous membrane to effect sample introduction into the capillary. In contrast, the present invention provides short disposable capillaries mounted in a frame that is integral with a liquid handling system. This system permits a rapid multiplexed approach to capillary electrophoresis.
Numerous examples of multi-well devices with integral membranes are known (e.g., Mann in U.S. Pat. No. 5,043,215, Matthis in U.S. Pat. No. 4,927,604, Bowers in U.S. Pat. No. 5,108,704, Clark in U.S. Pat. No. 5,219,528). Many of these devices attach to a base unit, which can be evacuated, drawing samples through the membrane for filtration.
Numerous examples of multi-channel metering devices such as multi channel pipettes are known. One example is described in a device by Schramm in U.S. Pat. No. 4,925,629, which utilizes an eight channel pipette to meter samples/reagents to/from multi-well plates. A second example is a 96 channel pipetting device described by Lyman in U.S. Pat. No. 4,626,509. These devices use positive displacement plungers in corresponding cylinders to draw in and expel liquid in the sampling/metering step.
Flesher in U.S. Pat. No. 5,213,766 describes a 96 channel device which contains flexible "fingers" which can be deformed out of a common plane; each "finger" can be deflected into a well of a multi-well plate to acquire a small aliquot of sample by one of several mechanisms.
Zare, et al., (U.S. Pat. No. 4,675,300) discusses a fluoroassay method for the detection of macromolecules such as genetic materials and proteins by capillary electrophoresis.
Yeung, et al., (U.S. Pat. No. 5,006,210) presented a system for capillary zone electrophoresis with indirect laser-induced fluorescence detection of macromolecules, including proteins, amino acids, and genetic materials. Systems such as these generally involve only one capillary.
U.S. Pat. Nos. 5,188,148 for a conduit plate for fluid delivery system and U.S. Pat. No. 5,325,889 for a laminated conduit plate for fluid delivery system both issued to Millipore Corp.
U.S. Pat. No. 5,463,910 discloses a multi-function aspirating device (AVL Scientific Corp.) U.S. Pat. No. 5,384,093 discusses an apparatus for aspirating and discharging a liquid sample (Toa Medical Electronics Co., Ltd.).
U.S. Pat. No. 5,525,302 discloses a method and device for simultaneously transferring plural samples.
A multiwell plate is disclosed in PCT WO 97/15394 published May 1, 1997 (SmithKline Beecham Corporation). The wells have a large opening at the top and small nozzle hole in the base. The opening is chosen so that a jet of liquid is emitted when a pressure pulse is applied to the surface such that by selecting a time for the pressure pulse a precise amount of volume in the well can be dispensed.